(a) Field of the Invention
The invention relates to chimeric fatty body-pro-GRF analogs with increased biological potency and prolonged activity, their application as anabolic agents and treatment of growth hormone deficiencies.
(b) Description of Prior Art
Growth hormone (GH) or somatotropin, secreted by the pituitary gland constitute a family of hormones which biological activity is fundamental for the linear growth of a young organism but also for the maintenance of the integrity at its adult state. GH acts directly or indirectly on the peripheral organs by stimulating the synthesis of growth factors (insulin-like growth factor-I or IGF-I) or of their receptors (epidermal growth factor or EGF). The direct action of GH is of the type referred to as anti-insulinic, which favors the lipolysis at the level of adipose tissues. Through its action on IGF-I (somatomedin C) synthesis and secretion, GH stimulate the growth of the cartilage and the bones (structural growth), the protein synthesis and the cellular proliferation in multiple peripheral organs, including muscles and the skin. Through its biological activity, GH participates within adults at the maintenance of a protein anabolism state, and plays a primary role in the tissue regeneration phenomenon after a trauma.
The decrease of GH secretion with the age, demonstrated in humans and animals, favors a metabolic shift towards catabolism which initiates or participate to the aging of an organism. The loss in muscle mass, the accumulation of adipose tissues, the bone demineralization, the loss of tissue regeneration capacity after an injury, which are observed in elderly, correlate with the decrease in the secretion of GH.
GH is thus a physiological anabolic agent absolutely necessary for the linear growth of children and which controls the protein metabolism in adults.
The secretion of GH by the pituitary gland is principally controlled by two hypothalamic peptides, somatostatin and growth hormone-releasing factor (GRF). Somatostatin inhibits its secretion, whereas GRF stimulates it.
The human GH has been produced by genetic engineering for about ten years. Until recently most of the uses of GH were concerned with growth delay in children and now the uses of GH in adults are studied. The pharmacological uses of GH and GRF may be classified in the following three major categories.
Children growth
Treatments with recombinant human growth hormone have been shown to stimulate growth in children with pituitary dwarfism, renal insufficiencies, Turner's syndrome and short stature. Recombinant human GH is presently commercialized as an "orphan drug" in Europe and in the United States for children's growth retardation caused by a GH deficiency and for children's renal insufficiencies. The other uses are under clinical trial investigation.
Long term treatment for adults and elderly patients
A decrease in GH secretion causes changes in body composition during aging. Preliminary studies of one-year treatment with recombinant human GH reported an increase in the muscle mass and in the thickness of skin, a decrease in fat mass with a slight increase in bone density in a population of aged patients. With respect to osteoporosis, recent studies suggest that recombinant human GH does not increase bone mineralization but it is suggested that it may prevent bone demineralization in post-menopausal women. Further studies are currently underway to demonstrate this theory.
Short term treatment in adults and elderly patients
In preclinical and clinical studies, growth hormone has been shown to stimulate protein anabolism and healing in cases of burn, AIDS and cancer, in wound and bone healing.
GH and GRF are also intended for veterinary pharmacological uses. Both GH and GRF stimulate growth in pigs during its fattening period by favoring the deposition of muscle tissues instead of adipose tissues and increase milk production in cows, and this without any undesired side effects which would endanger the health of the animals and without any residue in the meat or milk being produced. The bovine somatotropin (BST) is presently commercialized in the United States.
Most of the clinical studies presently undertaken were conducted with recombinant GH. The GRF is considered as a second generation product destined to replace in the near future the uses of GH in most instances. Accordingly, the use of GRF presents a number of advantages over the use of GH per se.
Physiological advantages
Growth hormone (GH) is secreted by the pituitary gland in a pulse fashion, since this rhythm of secretion is crucial for an optimal biological activity. The administration of GH to correspond to its natural mode of secretion is difficult to achieve. When GRF is administered in a continuous fashion as a slow releasing preparation or as an infusion, it increases GH secretion while respecting its pulsatility.
The recombinant GH which is presently commercialized is the 22 kDa form whereas GRF induces the synthesis and secretion from the pituitary gland of all the chemical isomers of GH which participate in a wider range of biological activities.
A treatment with GH results in a decreased capacity of the pituitary gland to secrete endogenous growth hormone, and the GH response to GRF is diminished after such a treatment. On the contrary, a treatment with GRF does not present this disadvantages, its trophic action on the pituitary gland increases this gland secreting capacity in normal animals and in patients with somatotroph insufficiency.
Economical advantages
The production of GH by genetic engineering is very expensive for clinical use. In particular, there are risks of contamination of these commercial preparation with material from the bacterial strain used. These bacterial contaminants may be pyrogens or may result in immunogenic reactions in patients. The purification of the recombinant product is effected by following a plurality of successive chromatography steps. The drastic purity criteria causes multiple quality control steps.
The synthesis of GRF is of chemical nature. The synthesis effected in a solid phase and its purification is carried out in a single step using high performance liquid chromatography (HPLC). Also the quantity of GRF to be administered is much less than the quantity of GH for the same resulting biological activity.
Even with all these advantages, GRF is still not commercialized to date as a therapeutic agent mainly because of its chemical instability. The human GRF is a peptide of 44 amino acids of the following sequence:
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln (SEQ ID NO:1) 1 5 10 15 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly 20 25 30 Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu--NH.sub.2. 35 40 The minimum active core is hGRF (1-29)NH.sub.2 Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln (SEQ ID NO:2) 1 5 10 15 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg. 20 25
As for many peptides, hGRF (1-29)NH.sub.2 is rapidly degraded in a serum medium and its metabolites have no residual biological activity. It has been well established that the action of enzymes, namely that of dipeptidylaminopeptidase type IV, in a blood medium results in the hydrolysis of the peptide bond Ala.sup.2 -Asp.sup.3 of GRF. This hydrolysis results in a multitude of negative consequences which was the subject of many studies reported in the literature. Essentially, this hydrolysis leads to the formation of truncated peptides of specific activity reduced to less than 1/1000 of the biological activity.
Clinical studies with children and adults have confirmed that natural hGRF (1-44)NH.sub.2 or the active fragment hGRF (1-29)NH.sub.2 are not potent enough to produce equal effects corresponding to those of recombinant GH.
Many GRF analogs have been described, but they all present the disadvantages of being modified GRP having a different amino acid sequence or having synthetic amino acids (D series) added. These GRP analogs are potentially immunogenic and their administration to human may cause immunotoxicity problems and potential side effects.
It is well known that the anchoring of hydrophobic groups, such as -NEt.sub.2 at the C-terminal of a peptidic sequence can result in a significantly increased specific activity. In terms of hydrophobicity, these results are contradicted by a fare number recent works such as those of Muranichi (S. Muranichi et al., 1991, Pharm. Res., 8:649-652) which stress the inefficacy of the lauroyl group as an hydrophobic group used in the synthesis of small peptides analogs. Hence, the contradictory investigations of the prior art failed to address the issue of finding a more potent GRP analog using hydrophobic residues.
Gaudreau et al. (P. Gaudreau et al., 1992, J. Med. Chem., 35(10),:1864-1869) describe the affinity of acetyl-, 6-aminohexanoyl-, and 8-aminooctanoyl-GRF(1-29)NH.sub.2 with the rat pituitary receptor. In this report, none of the fatty acid-GRF compounds tested exhibited a higher affinity than hGRF(1-29)NH.sub.2 itself, and the authors concluded that " . . . modifications to increase the hydrophobic character at the N-terminus of hGRF(1-29)NH.sub.2 do not constitute a suitable approach to increase receptor affinity".
Coy et al. (D. H. Cow et al., 1987, J. Med. Chem., 30:219-222) describe an acetyl-GRF peptide with an increased biological activity on a rat model, more particularly on a rat anesthetized with sodium pentobarbital. The in vitro GH response by cultured rat pituitary cells was also analyzed. However, these authors did not synthesize and test fatty acid-GRF analogs with a carbon chain longer than 2 (acetyl) added at the N-terminus region of the GRF.
Up to now, most of the GRF analogs described (including those of Gaudreau et al. and those of Coy et al.) have been tested in rat models, either in vitro or in vivo. Since human and rat GRF(1-29)NH.sub.2 are markedly different, the structure-activity relationships of GRF is different in both species. Therefore, it is not possible to extrapolate results obtained in rats to human.
Accordingly, it is necessary to design GRF analogs with improved anabolic potency and having a prolonged activity. This increased potency could result from a resistance to serum degradation and/or from hyperagonistic properties.
It would be highly desirable to be provided with GRF analogs with increased anabolic potency, while remaining biodegradable and structurally closed to natural GRF, in order to prevent immune reactions when chronically injected in humans and animals.